Abstract: The invention relates to archaeal pyrrolysyl tRNA synthetases lacking a nuclear localization signal and/or comprising a nuclear export signal. The invention also relates to polynucleotides encoding said pyrrolysyl tRNA synthetases, eukaryotic cells comprising said polynucleotide and tRNA acylated by the pyrrolysyl tRNA synthetase or a polynucleotide encoding such tRNA, methods utilizing said cells for preparing polypeptides comprising unnatural amino acid residues, and kits useful in said methods.

Abstract: The present invention is concerned with orthogonal translation systems which allow for the site-specific introduction of non-canonical amino acid residues into a target protein (POI) in a POI-mRNA-selective manner. Specifically, the present invention relates to assembler fusion proteins which bring an RNA-targeting polypeptide (RNA-TP) segment and an orthogonal aminoacyl tRNA synthetase (O-RS) segment into spatial proximity of one another, either by direct linkage in RNA-TP/O-RS fusion proteins, or though the action of “assemblers” fused to each of these segments in assembler fusion proteins (AFPs). The invention also relates to AFP combinations and nucleic acid molecules comprising a POI-encoding sequence together with a targeting nucleotide sequence that is able to interact with an RNA-TP.

Abstract: The invention relates to archaeal pyrrolysyl tRNA synthetases lacking a nuclear localization signal and/or comprising a nuclear export signal. The invention also relates to polynucleotides encoding said pyrrolysyl tRNA synthetases, eukaryotic cells comprising said polynucleotide and tRNA acylated by the pyrrolysyl tRNA synthetase or a polynucleotide encoding such tRNA, methods utilizing said cells for preparing polypeptides comprising unnatural amino acid residues, and kits useful in said methods.

Abstract: The invention relates to archaeal pyrrolysyl tRNA synthetases lacking a nuclear localization signal and/or comprising a nuclear export signal. The invention also relates to polynucleotides encoding said pyrrolysyl tRNA synthetases, eukaryotic cells comprising said polynucleotide and tRNA acylated by the pyrrolysyl tRNA synthetase or a polynucleotide encoding such tRNA, methods utilizing said cells for preparing polypeptides comprising unnatural amino acid residues, and kits useful in said methods.

Abstract: The invention relates to a method of preparing engineered target polypeptides (TP) comprising in its amino acid sequence one or more, identical or different, non-canonical amino acid (ncAA) residues, by expressing said TP in an insect cell line (ICL) and by expressing novel orthogonal bacterial aminoacyl tRNA synthetase/tRNA pairs in said ICL; a baculoviral shuttle vector (bacmid) suitable or introducing the genetic information of said orthogonal tRNA synthetase/tRNA into said ILC; particular expression cassettes for expressing said particular tRNAs in said ILC; TPs obtained by said method; as we as a kit for preparing said TPs.